The invention relates to novel tubulin depolymerization agents, SPIKET, as potent anti-cancer agents.
Cancer is a major disease that continues as one of the leading causes of death at any age. In the United States alone, it is anticipated that more than a half a million Americans will die of cancer in 1999. Currently, radiotherapy and chemotherapy are two important methods used in the treatment of cancer.
Considerable efforts are underway to develop new chemotherapeutic agents for more potent and specific anti-cancer therapy, presenting effective and efficient cytotoxicity against tumor cells, with minimal interference with normal cell function. Accordingly, there is an urgent need for the development and analysis of novel, effective anti-cancer agents.
Cellular proliferation, for example, in cancer and other cell proliferative disorders, occurs as a result of cell division, or mitosis. Microtubules play a pivotal role in mitotic spindle assembly and cell division1-5. These cytoskeletal elements are formed by the self-association of the ad tubulin heterodimers1-5. Agents which induce depolymerization of tubulin and/or inhibit the polymerization of tubulin provide a therapeutic approach to the treatment of cell proliferation disorders such as cancer.
Recently, the structure of the xcex1xcex2 tubulin dimer was resolved by electron crystallography of zinc-induced tubulin sheets6. According to the reported atomic model, each 46xc3x9740xc3x9765 xc3x85 tubulin monomer is made up of a 205 amino acid N-terminal GTP/GDP binding domain with a Rossman fold topology typical for nucleotide-binding proteins, a 180 amino acid intermediate domain comprised of a mixed xcex2 sheet and five helices which contain the taxol binding site, and a predominantly helical C-terminal domain implicated in binding of microtubule-associated protein (MAP) and motor proteins2, 5.
Novel tubulin-binding molecules which, upon binding to tubulin, interfere with tubulin polymerization, can provide novel agents for the inhibition of cellular proliferation and treatment of cancer.
Spongistatin (SP) (FIG. 1) is a potent tubulin depolymerizing natural product isolated from an Eastern Indian Ocean sponge in the genus Spongia7. Spongistatins are 32-membered macrocyclic lactone compounds with a spongipyran ring system containing 4 pyran-type rings incorporated into two spiro[5.5]ketal moieties7. In cytotoxicity assays, spongistatin (SP) exhibited potent cytotoxicity with subnanomolar IC50 values against an NCI panel of 60 human cancer cell lines7. SP was found to inhibit the binding of vinc alkaloids (but not colchicin) to tubulin8, indicating that the binding site for this potent tubulin depolymerizing agent may also serve as a binding region for vinc alkaloids.
Novel tubulin binding compounds, which, upon binding to tubulin, interfere with tubulin assembly, for example by causing depolymerization of tubulin or by inhbiting tubulin polymerization, would provide novel agents for the prevention of cellular proliferation, for example in the inhibition of tumor cell growth and treatment of cancer.
A novel binding pocket has been identified in tubulin, which binding pocket accepts and binds novel, small molecule tubulin binding spiroketal pyrane compounds of the invention. Binding of the spiroketal pyranes (SPIKETs) to tubulin causes tubulin depolymerization, and/or inhibits tubulin polymerization. The siroketal pyranes of the invention are therapeutically effective as cytotoxic agents, to inhibit cellular proliferation, and as effective anti-cancer agents.
The compounds of the invention have the general structure shown in formula I, and are designed to include moieties and/or substitutions capable of favorable interaction with amino acid residues in the SPIKET binding pocket of tubulin. For example, R1, R2, S1, and S2 preferably contain functional groups and/or substitutions designed to favor hydrophobic interaction and/or Van der Waals interaction with hydrophobic residues of the SPIKET binding pocket, as described more fully below. 
wherein
X1, X2, and X3 are the same or different, and are each independently O, C, or S;
R1 and R2 are the same or different and are each independently H, provided both R1 and R2 are not H, or (C1-C8)alkyl, (C1-C8)cycloalkyl, (C1-C8)alkoxy, (C1-C8)aryloxy, (C1-C8)arylthio, (C1-C8)aryl, (C1-C8)heteroaryl, C(xe2x95x90)NRaRb or NRaRb; wherein Ra and Rb are each independently hydrogen, acyl, (C1-C8)alkyl, (C3-C7)cycloalkyl, (C6-C10)aryl, or (C6-C10)heteroaryl, or Ra and Rb together with the nitrogen to which they are attached form a ring such as pyrrolidino, piperidino, morpholino, or thiomorpholino;
n and m are the same or different, and are each independently 0 to 7;
S1 and S2 can be the same or different, and are each independently OH, SH, CO2H, halogen, CN, acyl, thioacyl, ester, thioester, (C1-C6)alkoxy, (C1-C6)aryloxy, (C1-C6)alkylthio, (C1-C6)arylthio, (C1-C6)alkyl, (C1-C6)alkenyl, (C1-C6)alkynyl, (C3-C7)cycloalkyl, (C6-C10)aryl, or (C6-C10)heteroaryl, C(xe2x95x90O)NRaRb or NRaRb; wherein Ra and Rb are each independently hydrogen, acyl, (C1-C6)alkyl, (C3-C7)cycloalkyl, (C6-C10)aryl, or (C6-C10)heteroaryl, or Ra and Rb together with the nitrogen to which they are attached form a ring such as pyrrolidino, piperidino, morpholino, or thiomorpholino; taken together, any two S1 and S2 can form a ring, and any two adjacent substituents can form a double bond between the two carbons to which they are attached. The hydrocarbon moieties of R1, R2, S1, and S2 may be substituted or unsubstituted.
R1, R2, Ra, Rb, S1, and S2 can be substituted with at least one functional group selected from OH, SH, CO2H, halogen, CN, acyl, thioacyl, ester, thioester, alkoxy, or aryloxy. In some embodiments, R1, R2, Ra, Rb, S1, and S2 comprise (C1-C8)alkyl groups substituted with OH, an ester, an alkoxy, or an aryloxy group. For example, R1, R2, Ra, Rb, S1, and S2 comprise xe2x80x94(CH2)nOH, n being 0-7, 
wherein a benzene ring can be substituted with a methoxy, halo, methyl, trifluoromethyl, or nitro.
Particular embodiments of the claimed compounds, for example, those compounds depicted in formulae II-V and preferred compounds of the invention are described below in the Detailed Description, Examples, and Claims. Particularly preferred compounds of the invention are:
[(2R,8R)-8-(hydroxymethyl)-1,7-dioxaspiro[5,5]undec-2-yl]methan-1-ol(SPIKET-P1);
Benzyl-protected SPIKET-P1 (SPIKET-P1-P); and
1,13-dibenzyloxy-5,9-dihydroxy-spiroketal (NP25).
The compounds of the invention are combined with a suitable carrier to form compositions suitable for use in binding tubulin, inducing depolymerization of tubulin, inhibiting proliferation of cells, and in the treatment of cancer.
The SPIKET binding pocket on tubulin is useful for designing and screening tubulin binding molecules, anti-cell proliferation agents, and cancer therapeutic agents. Useful agents are designed to fit the pocket and to favorably interact with the pocket for enhanced binding and anti-tubulin activity.
Additional embodiments of the invention are described more fully below.